Phenylketonuria (PKU) is a severely handicapping disorder if not diagnosed and treated early in life. PKU is a human genetic disorder caused by an inborn error in aromatic amino acid metabolism. It was first observed some 50 years ago that patients with this condition had excess phenylpyruvic acid in the urine (Folling A., 1934 Nord. Med. Tidskr8, 1054-1059; Folling, A., 1934 B, Zitschr. Physiol. Chem. 227, 169-176, 1934). Subsequently, it was discovered that livers of such which is the major metabolic pathway of phenylalanine (Jervis, G. A. J. Biol. Chem. 169, 651-656, 1947). Clinically, untreated PKU patients present with mental retardation, pigment dilution, hyperkinesis, microcephaly, and seizure activity. However, with the establishment of mass neonatal screening programs (Guthrie and Suzi, Pediat 32, 33-34, 1963) and the institution of early dietary therapy (Bickel, H. et al, Acta Pediat. Scand 43, 64-77, 1954), the classical clinical presentation is becoming a medical rarity in Western countries. Since mass screening occasionally misses an affected child, follow-up tests of newborns are important. Heterozygote detection followed by genetic counselling of individuals and families with PKU children should guarantee that future children within the families receive these follow-up tests.
Classical PKU is characterized by a lack of phenylalanine hydroxylase activity in the liver. The lack of this enzymatic activity causes persistent hyperphenylalaninemia, resulting in the minor metabolic pathways for phenylalanine becoming over utilized. High levels of phenylalanine and/or its derivatives are toxic and cause disturbances in tyrosine and tryptophan metabolism. Diminished formation of catecholamines, melanin and seratonin is typical in individuals with phenylalanine hydroxylase deficiency. In addition, the melanin sheath surrounding neuronal axons is not properly formed in the brains of untreated PKU patients and the clinical symptoms described above are irreversible.
Phenylketonuria patients secrete large quantities of phenylpyruvate in the urine which can be readily detected by its reaction with ferric chloride. This reaction was used in the 1950's as a screening test for the diagnosis of PKU children. However, in 1962, a simple mass screening method for a semi-quantitative determination of phenylalanine in small samples of blood for newborn infants was subsequently developed by Guthrie and Suzi (Pediat. 32, 33-343, 1963). However, individuals diagnosed and treated for classical PKU do not necessarily achieve a normal I.Q. Initiation of dietary therapy soon after birth results in a major disruption of lifestyle and has tremendous psychological and social implications for the patient's family. Dietary regulation must be implemented over a prolonged period of time to be effective and elimination of treatment in the mid first decade is apparently followed by a small but significant decline in I.Q. scores. The supervision of this treatment is difficult and is best performed only at centers experienced with such regimens.
Phenylalanine hydroxylase deficiency, regardless of phenotypic variations, is transmitted as an autosomal recessive trait. Mass screening of over 5,000,000 neonates has shown that the prevalance of classical phenylketonuria in Caucasions ranges from 1/5000 to 1/16000. The collective frequency in western Europe and the United States is about 1/8000 resulting in 2% of the population being heterozygote carriers of the PKU trait.
Many investigators have recognized the need for heterozygosity testing. These tests have involved a variety of methods including using oral phenylalanine loading tests, fasting blood samples in the early morning, semi-fasting mid-day samples, and sophisticated statistical methods for discriminating between heterozygotes and normals. None of the tests that have been developed have a 100% accuracy. All of the tests show some theoretical overlap between heterozyqote and homozygote normal individuals. The main problem is that the measurement of blood phenylalanine is in reality a measurement of secondary phenomena. The enzyme that is deficient, phenylalanine hydroxylase, is only present in the liver. Very few people are willing to give liver biopsies to determine their genetic constitution. Stable isotope analysis is another method used for indirect measurements of this enzyme activity. Phenylalanine is labelled with a non-radioactive .sup.13 C or .sup.2 H and then phenylalanine and its metabolites are measured by mass spectrometry. Again, this is not a direct assay since it measures not only the reaction and conversion of phenylalanine to tyrosine but also the exchange and transport of phenylalanine and tyrosine in the whole body.
With the emergence of molecular biological technology, new methods became available to measure the gene responsible for phenylalanine hydroxylase deficiency and to determine the heterozygosity or genetic state of an individual (Woo, et. al., Nature 306, 151-155, 1983, Lidsky, et. al., Am. J. Human Genet. 37, 619-634, 1985, and Patent Application Ser. Nos. 484,816 and its continuation-in-part Ser. No. 600,254). These methods of heterozygote detection require the ascertainment of a family through a proband. Furthermore, they require family studies in order to determine the segregation of the PKU alleles with restriction fragment length polymorphisms (RFLP) at the phenylalanine hydroxylase (PAH) locus. Even with family studies, the extensive RFLP's identified in the human phenylalanine hydroxylase locus still leaves some families without a method for detection of heterozygosity or for prenatal diagnosis of affected PKU individuals. The present invention is directed to a new and improved use of molecular biological technology to measure the actual mutations in the PAH locus.
Hydroxylation of phenylalanine to tyrosine involves a complex enzyme system involving at least two enzymes directly and numerous cofactors. Experience over the years has taught that the deficiency exists in more than just the classical PKU state. There is tremendous heterogeniety among the phenotypes and in the hydroxylase deficiency. These include hyperphenylalaninamia, enzyme cofactor deficiencies specifically in the enzyme of dihydropteridine reductase, and cofactor deficiencies. These types of "atypical phenyketonuria" however constitute only a minor percentage of the PKU patients observed. A vast majority of the PKU patients result from a deficiency in the liver enzyme phenylalanine hydroxylase. However, phenylalanine hydroxylase deficiency itself is very heterogenous resulting in diseases ranging from severe, classical PKU, to moderate, hyperphenylalaninemia, states.
The enzyme has been isolated and characterized. It is a multimeric enzyme with a subunit molecular weight of approximately 50,000 daltons. Recent evidence has shown that the subunits are either identical or are precursor - product of one another (Ledley, et. al. Science 228 77-79, 1985). In some patients with classical PKU, PAH has been found to exist in a structurally altered form with less than 1% of the normal activity (Bartholome, et al, Pediat Res. 9, 899-903, 1975), indicating PKU may be the direct result of a mutation in a PAH gene itself. Furthermore, analysis of PAH activity in liver biopsies of individuals with hyperphenylalaninemia demonstrated that the enzyme contained about 5% of the normal activity level with a range of 2 to 35%. The evidence indicates that the low enzymatic activities of hyperphenylalaninemics are not due to the presence of 5% of the normal enzyme, but rather to the presence of an altered PAH with kinetic properties distinct from both the normal enzyme and the enzyme from patients with classical PKU. These results clearly indicate the presence of multiple phenotypes of PAH deficiency, probably resulting from mutations at various sites in the PAH gene locus.
Phenylalanine hydroxylase cDNA clones have been isolated from rat and human liver cDNA libraries. Our earlier patent applications, Ser. No. 484,816, and Ser. No. 600,254, as well as the publication in Nature 306, 151-155, 1983 disclose the use of human PAH cDNA clones representing the 3' half of the PAH messenger RNA(mRNA), to identify RFLP's in the PAH locus in man. The polymorphisms were found using the restriction enzymes MspI, SphI and HindIII and have been applied to trace the transition of the mutant PAH genes in informative PKU families with one or more affected children. These results demonstrated that the mutant PAH genes segregated concordantly with the disease state. The frequencies of the restriction site polymorphism in the PAH gene detected by the partial cDNA clones are such that even under ideal conditions only 70% of the PKU families in the population can take advantage of the genetic analysis. Later studies, patent application Ser. No. 600,254 Kwok, et al, Biochemistry 24, 556-561, 1985 and Lidsky, et. al. Am. J. Human Genet. 37, 619-634, 1985, describe nucleotide sequence use of a full-length human PAH cDNA clone. Although this significantly increased the number of RFLPs, some families were still unable to utilize this type of genetic analysis.
The new full-length cDNA probe detects six RFLP's that were not previously detected with the 3'-PAH cDNA probe. These additional polymorphisms, representing EcoRI, BglII, XmnI, EcoRV, PvuII(a) and PvuII(b), are mapped and reside within the PAH gene or in the immediate flanking genomic sequences (Di Lella, et. al., Biochem 25, 743-749, 1986). The 8 polymorphic restriction enzyme sites in the PAH locus were analyzed in 33 Danish families with at least 1 PKU child. The studies in the Danish population showed a high variability in the number of haplotypes, 12 were found. However, this represents a small number compared to a theoretical value of 1,936 haplotypes. Thus, there is strong evidence indicating a tight linkage of the RFLP's within the gene itself and the presence of linkage disequilibrium. Even with the increased amount of variability, 87%, found in the Danish population with the full-length cDNA and the restriction endonucleases (Daiger et. al., Lancet I, 229-232, 1986), there still exist families which cannot utilize this genetic technique. Additionally, the technique has the disadvantage of requiring the ascertainment of a PKU proband before the family can utilize the method. To circumvent these problems, investigations were instituted to identify the specific mutations and to develop methods which detect the specific mutations resulting in altered PAH genes.